Abstract
The CCR7 ligands CCL19 and CCL21 are increasingly recognized as functionally different (biased). Using mature human dendritic cells (DCs), we show that CCL19 is more potent than CCL21 in inducing 3D chemotaxis. Intriguingly, CCL21 induces prolonged and more efficient ERK1/2 activation compared with CCL19 and a C-terminal truncated (tailless) CCL21 in DCs. In contrast, tailless-CCL21 displays increased potency in DC chemotaxis compared with native CCL21. Using a CCL21-specific antibody, we show that CCL21, but not tailless-CCL21, accumulates at the cell surface. In addition, removal of sialic acid from the cell surface by neuraminidase treatment impairs ERK1/2 activation by CCL21, but not by CCL19 or tailless-CCL21. Using standard laboratory cell lines, we observe low potency of both CCL21 and tailless-CCL21 in G protein activation and β-arrestin recruitment compared with CCL19, indicating that the tail itself does not improve receptor interaction. Chemokines interact with their receptors in a stepwise manner with ultimate docking of their N-terminus into the main binding pocket. Employing site-directed mutagenesis we identify residues in this pocket of selective CCL21 importance. We also identify a molecular switch in the top of TM7 important for keeping CCR7 in an inactive conformation (Tyr312), as introduction of the chemokine receptor-conserved Glu (or Ala) induces high constitutive activity. Summarized, we show that the interaction of the tail of CCL21 with polysialic acid is needed for strong ERK signaling, whereas it impairs CCL21-mediated chemotaxis and has no impact on receptor docking consistent with the current model of chemokine:receptor interaction. This indicates that future selective pharmacological targeting of CCL19 versus CCL21 should focus on a differential targeting of the main receptor pocket, while selective targeting of tailless-CCL21 versus CCL21 and CCL19 requires targeting of the glycosaminoglycan (GAG) interaction.
Highlights
Chemokines are chemotactic cytokines that are vital for the immune system, especially as mediators of immune cell recruitment to sites of inflammation
As migration has classically been linked to ERK activation, we investigate the effect of CCL19 and CCL21 on CCR7 signaling through this pathway in dendritic cells (DCs) and assess the influence of sialic acid residues on ERK activity and migration
To assess the relative importance of the two CCR7 ligands in stimulating various DC responses, we investigated the effect of CCL19 and CCL21 on a range of biological readouts
Summary
Chemokines are chemotactic cytokines that are vital for the immune system, especially as mediators of immune cell recruitment to sites of inflammation. Bias of Three CCR7 Chemokines in DCs are organized into groups according to the spacing between two conserved cysteine residues. This gives rise to four groups: CC, CXC, XC, and CX3C chemokines, with the number of Xs’ indicating the number of amino acids separating the conserved cysteines. The chemokines CCL19 and CCL21 [previously known as EBI1-ligand chemokine (ELC) and secondary lymphoid tissue chemokine (SLC), respectively] are the only ligands for CCR7 [1,2,3]; a receptor expressed on different subsets of immune cells [4], and involved in the homing of naive T cells and antigenpresenting dendritic cells (DCs) to the lymph nodes. In addition to its role in protective immunity, the CCR7:CCL19/CCL21 axis is believed to be important for the architecture of the thymus [6, 7] and for the induction of peripheral tolerance [5, 8]
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